Human tumor necrosis factor-α (TNFα) is a proinflammatory cytokine produced by monocytes and macrophages, which is a 26 kDA precursor protein when initially produced with N terminal inside the cells and C terminal outside the cells, named transmembrane type TNFα. Pennica et al. cloned TNFα gene cDNA for the first time in 1984, and deduced that human TNFα molecule is composed of 157 amino acid residues, and weights about 17 KD (Pennica D, et al, Nature 1984; 312:724). Huamn TNF has two molecular forms, TNFα and TNFβ. TNFα is produced by activated macrophages or monocytes, and causes neoplastic tissues hengrrhagie necrosis, thus it is also called Cachectin. TNFβ is mainly secreted by active T lymphocytes. Both have similar pyretogenesis. TNFα acts on receptors on the surface of oncocytes, and breaks into lysosome by identifying the cell, binding and endocytosing, and then activates lysosomes and proteases to cause cell death. TNFα plays an important role in immune response, inflammation, and response to injury, majorly affects the regulation of cell proliferation and cell apoptosis. Besides the effects on tumor cell such as cytotoxicity, cytolysis, induction of apoptosis and cell proliferation suppression, TNFα also can facilitate cell differentiation of myeloid leukemia cell to macrophage, and improve the phagocytic activity of neutrophile granulocyte.
An appropriate amount of TNFα can activate immune system to enhance immunity of the body, and play an important role in defense system of host resisting microbial invasion and tumor inhibition. But when over expressed, TNFα may cause several pathologic damages with other inflammatory factors. Therefore, the activities of TNFα may be suppressed or neutralized at different levels to block it from approaching receptors, in turn avoid the consequence of signal transduction.
For the purpose of overcoming relevant problems caused by using non-human antibodies, it is a relatively effective strategy of treatment to construct human-murine chimeric antibody to decrease organism immunogenicity initiated by HAMA. Such kind of chimeric antibody is made by incorporating non-human antibody variable region into human antibody constant region while retaining amino acid sequences of original heavy chain, light chain variable regions of non-human antibody (see, Daddona, P. E et al. PCT publication WO92/16553, Le, J. et al., U.S. Pat. No. 5,919,452, Kang, Heui H et al. PCT publication WO2005/047329, Jin BOquan et al. Chinese patent publication CN1544466A), Jin Yihui et al. Chinese patent publication CN101177453 provides a chimeric antibody which can bind to human tumor necrosis factor. Compared to non-human antibody, the immunogenicity of the chimeric antibody decreases, however, it may cause HAMA response in varied degrees since the murine derived portion in the chimeric antibody is still high, specifically including skin mucosa reaction, allergic reaction, arrhythmia and stenocardia, renal insufficiency, even coma when severe. Therefore, the clinical applications of this kind of chimeric antibodies are greatly limited.
Clinical trials demonstrate that this kind of chimeric antibodies as heterogeneous protein may cause immunological rejecting response of the heterogeneous protein by organism immune system (i.e., Human anti mouse antibody, HAMA response) when administrated to human. The response leads to rapid clearance of the murine antibody in human bodies, and short half life. Repeated administration may even result in severe anaphylactic shock. Moreover, the “foreign” antibody may be attacked by immune antibody, so that they may be neutralized before presenting pharmaceutical effects.
The inventors develop a new technique to prepare humanized antibody by utilizing genetic technology for the purpose of reducing the murine derived portion to minimum in the chimeric antibody on the basis of aforesaid patents. The technique comprises separately incorporating complementary determining regions (CDRs) of murine antibody heavy chain variable region and light chain variable region into human antibody framework region (FR). The obtained humanized antibody is similar to human sequence in structure as possible, meantime, it also can maintain CDR conformation similar to parent non-human antibody. Compared to parent non-human antibody and chimeric antibody, the portion of parent non-human amino acid sequence in the engineered humanized antibody decreases, one hand, the ability of antibody recognizing antigen is remained; the other hand, the immunogenicity of murine antibody has been greatly decreased. Therefore, safety of the antibody in clinical applications has been improved.
Consequently, the present invention provides a humanized antibody, which is safer, has longer half life more significant effects in human body, compared to murine chimeric antibody in prior arts.